A plant's traits, such as its biochemical, developmental, or phenotypic characteristics, may be controlled through a number of cellular processes. One important way to manipulate that control is through transcription factors—proteins that influence the expression of a particular gene or sets of genes. Transformed and transgenic plants that comprise cells having altered levels of at least one selected transcription factor, for example, possess advantageous or desirable traits. Strategies for manipulating traits by altering a plant cell's transcription factor content can therefore result in plants and crops with new and/or improved commercially valuable properties.
Transcription factors can modulate gene expression, either increasing or decreasing (inducing or repressing) the rate of transcription. This modulation results in differential levels of gene expression at various developmental stages, in different tissues and cell types, and in response to different exogenous (e.g., environmental) and endogenous stimuli throughout the life cycle of the organism.
Because transcription factors are key controlling elements of biological pathways, altering the expression levels of one or more transcription factors can change entire biological pathways in an organism. For example, manipulation of the levels of selected transcription factors may result in increased expression of economically useful proteins or biomolecules in plants or improvement in other agriculturally relevant characteristics. Conversely, blocked or reduced expression of a transcription factor may reduce biosynthesis of unwanted compounds or remove an undesirable trait. Therefore, manipulating transcription factor levels in a plant offers tremendous potential in agricultural biotechnology for modifying a plant's traits. A number of the agriculturally relevant characteristics of plants, and desirable traits that may be imbued by modified transcription factor gene expression, are listed below.
Useful Plant Traits
Category: Abiotic Stress; Desired Trait: Chilling Tolerance
The term “chilling sensitivity” has been used to describe many types of physiological damage produced at low, but above freezing, temperatures. Most crops of tropical origins such as soybean, rice, maize and cotton are easily damaged by chilling. Typical chilling damage includes wilting, necrosis, chlorosis or leakage of ions from cell membranes. The underlying mechanisms of chilling sensitivity are not completely understood yet, but probably involve the level of membrane saturation and other physiological deficiencies. For example, photoinhibition of photosynthesis (disruption of photosynthesis due to high light intensities) often occurs under clear atmospheric conditions subsequent to cold late summer/autumn nights. By some estimates, chilling accounts for monetary losses in the United States (US) second only to drought and flooding. For example, chilling may lead to yield losses and lower product quality through the delayed ripening of maize. Another consequence of poor growth is the rather poor ground cover of maize fields in spring, often resulting in soil erosion, increased occurrence of weeds, and reduced uptake of nutrients. A retarded uptake of mineral nitrogen could also lead to increased losses of nitrate into the ground water.
Category: Abiotic Stress; Desired Trait: Freezing Tolerance.
Freezing is a major environmental stress that limits where crops can be grown and that reduces yields considerably, depending on the weather in a particular growing season. In addition to exceptionally stressful years that cause measurable losses of billions of dollars, less extreme stress almost certainly causes smaller yield reductions over larger areas to produce yield reductions of similar dollar value every year. For instance, in the US, the 1995 early fall frosts are estimated to have caused losses of over one billion dollars to corn and soybeans. The spring of 1998 saw an estimated $200 M of damages to Georgia alone, in the peach, blueberry and strawberry industries. The occasional freezes in Florida have shifted the citrus belt further south due to $100 M or more losses. California sustained $650 M of damage in 1998 to the citrus crop due to a winter freeze. In addition, certain crops such as Eucalyptus, which has the very favorable properties of rapid growth and good wood quality for pulping, are not able to grow in the southeastern states due to occasional freezes.
Inherent winter hardiness of the crop determines in which agricultural areas it can survive the winter. For example, for wheat, the northern central portion of the US has winters that are too cold for good winter wheat crops. Approximately 20% of the US wheat crop is spring wheat, with a market value of $2 billion. Areas growing spring wheat could benefit by growing winter wheat that had increased winter hardiness. Assuming a 25% yield increase when growing winter wheat, this would create $500 M of increased value. Additionally, the existing winter wheat is severely stressed by freezing conditions and should have improved yields with increased tolerance to these stresses. An estimate of the yield benefit of these traits is 10% of the $4.4 billion winter wheat crop in the US or $444 M of yield increase, as well as better survival in extreme freezing conditions that occur periodically.
Thus plants more resistant to freezing, both midwinter freezing and sudden freezes, would protect a farmers' investment, improve yield and quality, and allow some geographies to grow more profitable and productive crops. Additionally, winter crops such as canola, wheat and barley have 25% to 50% yield increases relative to spring planted varieties of the same crops. This yield increase is due to the “head start” the fall planted crop has over the spring planted crop and its reaching maturity earlier while the temperatures, soil moisture and lack of pathogens provide more favorable conditions.
Category: Abiotic Stress; Desired Trait: Salt Tolerance.
One in five hectares of irrigated land is damaged by salt, an important historical factor in the decline of ancient agrarian societies. This condition is only expected to worsen, further reducing the availability of arable land and crop production, since none of the top five food crops—wheat, corn, rice, potatoes, and soybean—can tolerate excessive salt.
Detrimental effects of salt on plants are a consequence of both water deficit resulting in osmotic stress (similar to drought stress) and the effects of excess sodium ions on critical biochemical processes. As with freezing and drought, high saline causes water deficit; the presence of high salt makes it difficult for plant roots to extract water from their environment (Buchanan et al. (2000) in Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, Rockville, Md.). Soil salinity is thus one of the more important variables that determines where a plant may thrive. In many parts of the world, sizable land areas are uncultivable due to naturally high soil salinity. To compound the problem, salination of soils that are used for agricultural production is a significant and increasing problem in regions that rely heavily on agriculture. The latter is compounded by over-utilization, over-fertilization and water shortage, typically caused by climatic change and the demands of increasing population. Salt tolerance is of particular importance early in a plant's lifecycle, since evaporation from the soil surface causes upward water movement, and salt accumulates in the upper soil layer where the seeds are placed. Thus, germination normally takes place at a salt concentration much higher than the mean salt level in the whole soil profile.
Category: Abiotic Stress; Desired Trait: Drought Tolerance.
While much of the weather that we experience is brief and short-lived, drought is a more gradual phenomenon, slowly taking hold of an area and tightening its grip with time. In severe cases, drought can last for many years, and can have devastating effects on agriculture and water supplies. With burgeoning population and chronic shortage of available fresh water, drought is not only the number one weather related problem in agriculture, it also ranks as one of the major natural disasters of all time, causing not only economic damage, but also loss of human lives. For example, losses from the US drought of 1988 exceeded $40 billion, exceeding the losses caused by Hurricane Andrew in 1992, the Mississippi River floods of 1993, and the San Francisco earthquake in 1989. In some areas of the world, the effects of drought can be far more severe. In the Horn of Africa the 1984-1985 drought led to a famine that killed 750,000 people.
Problems for plants caused by low water availability include mechanical stresses caused by the withdrawal of cellular water. Drought also causes plants to become more susceptible to various diseases (Simpson (1981). “The Value of Physiological Knowledge of Water Stress in Plants”, In Water Stress on Plants, (Simpson, G. M., ed.), Praeger, N.Y., pp. 235-265).
In addition to the many land regions of the world that are too arid for most if not all crop plants, overuse and over-utilization of available water is resulting in an increasing loss of agriculturally-usable land, a process which, in the extreme, results in desertification. The problem is further compounded by increasing salt accumulation in soils, as described above, which adds to the loss of available water in soils.
Category: Abiotic Stress; Desired Trait: Heat Tolerance.
Germination of many crops is very sensitive to temperature. A transcription factor that would enhance germination in hot conditions would be useful for crops that are planted late in the season or in hot climates.
Seedlings and mature plants that are exposed to excess heat may experience heat shock, which may arise in various organs, including leaves and particularly fruit, when transpiration is insufficient to overcome heat stress. Heat also damages cellular structures, including organelles and cytoskeleton, and impairs membrane function (Buchanan, supra).
Heat shock may result a decrease in overall protein synthesis, accompanied by expression of heat shock proteins. Heat shock proteins function as chaperones and are involved in refolding proteins denatured by heat.
Category: Abiotic Stress; Desired Trait: Tolerance to Low Nitrogen and Phosphorus.
The ability of all plants to remove nutrients from their environment is essential to survival. Thus, identification of genes that encode polypeptides with transcription factor activity may allow for the generation of transgenic plants that are better able to make use of available nutrients in nutrient-poor environments.
Among the most important macronutrients for plant growth that have the largest impact on crop yield are nitrogenous and phosphorus-containing compounds. Nitrogen- and phosphorus-containing fertilizers are used intensively in agriculture practices today. An increase in grain crop yields from 0.5 to 1.0 metric tons per hectare to 7 metric tons per hectare accompanied the use of commercial fixed nitrogen fertilizer in production farming (Vance (2001) Plant Physiol. 127: 390-397). Given current practices, in order to meet food production demands in years to come, considerable increases in the amount of nitrogen- and phosphorus-containing fertilizers will be required (Vance, supra).
Nitrogen is the most abundant element on earth yet it is one of the most limiting elements to plant growth due to its lack of availability in the soil. Plants obtain N from the soil from several sources including commercial fertilizers, manure and the mineralization of organic matter. The intensive use of N fertilizers in present agricultural practices is problematic, the energy intensive Haber-Bosch process makes N fertilizer and it is estimated that the US uses annually between 3-5% of the nation's natural gas for this process. In addition to the expense of N fertilizer production and the depletion of non-renewable resources, the use of N fertilizers has led to the eutrophication of freshwater ecosystems and the contamination of drinking water due to the runoff of excess fertilizer into ground water supplies.
Phosphorus is second only to N in its importance as a macronutrient for plant growth and to its impact on crop yield. Phosphorus (P) is extremely immobile and not readily available to roots in the soil and is therefore often growth limiting to plants. Inorganic phosphate (Pi) is a constituent of several important molecules required for energy transfer, metabolic regulation and protein activation (Marschner (1995) Mineral Nutrition of Higher Plants, 2nd ed., Academic Press, San Diego, Calif.). Plants have evolved several strategies to help cope with P and N deprivation that include metabolic as well as developmental adaptations. Most, if not all, of these strategies have components that are regulated at the level of transcription and therefore are amenable to manipulation by transcription factors. Metabolic adaptations include increasing the availability of P and N by increasing uptake from the soil though the induction of high affinity and low affinity transporters, and/or increasing its mobilization in the plant. Developmental adaptations include increases in primary and secondary roots, increases in root hair number and length, and associations with mycorrhizal fungi (Bates and Lynch (1996) Plant Cell Environ. 19: 529-538; Harrison (1999) Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 361-389).
Category: Biotic Stress; Desired Trait: Disease Resistance.
Disease management is a significant expense in crop production worldwide. According to EPA reports for 1996 and 1997, us farmers spend approximately $6 billion on fungicides annually. Despite this expenditure, according to a survey conducted by the food and agriculture organization, plant diseases still reduce worldwide crop productivity by 12% and in the United States alone, economic losses due to plant pathogens amounts to 9.1 billion dollars (FAO, 1993). Data from these reports and others demonstrate that despite the availability of chemical control only a small proportion of the losses due to disease can be prevented. Not only are fungicides and anti-bacterial treatments expensive to growers, but their widespread application poses both environmental and health risks. The use of plant biotechnology to engineer disease resistant crops has the potential to make a significant economic impact on agriculture and forestry industries in two ways: reducing the monetary and environmental expense of fungicide application and reducing both pre-harvest and post-harvest crop losses that occur now despite the use of costly disease management practices.
Fungal, bacterial, oomycete, viral, and nematode diseases of plants are ubiquitous and important problems, and often severely impact yield and quality of crop and other plants. A very few examples of diseases of plants include:
Powdery mildew, caused by the fungi Erysiphe, Sphaerotheca, Phyllactinia, Microsphaera, Podosphaera or Uncinula, in, for example, wheat, bean, cucurbit, lettuce, pea, grape, tree fruit crops, as well as roses, phlox, lilacs, grasses, and Euonymus;
Fusarium-caused diseases such as Fusarium wilt in cucurbits, Fusarium head blight in barley and wheat, wilt and crown and root rot in tomatoes;
Sudden oak death, caused by the oomycete Phytophthora ramorum; this disease was first detected in 1995 in California tan oaks. The disease has since killed more than 100,000 tan oaks, coast live oaks, black oaks, and Shreve's oaks in coastal regions of northern California, and more recently in southwestern Oregon (Roach (2001) National Geographic News, Dec. 6, 2001);
Black Sigatoka, a fungal disease caused by Mycosphaerella species that attacks banana foliage, is spreading throughout the regions of the world that are responsible for producing most of the world's banana crop;
Eutypa dieback, caused by Eutypa lata, affects a number of crop plants, including vine grape. Eutypa dieback delays shoot emergence, and causes chlorosis, stunting, and tattering of leaves;
Pierce's disease, caused by the bacterium Xylella fastidiosa, precludes growth of grapes in the southeastern United States, and threatens the profitable wine grape industry in northern California. The bacterium clogs the vasculature of the grapevines, resulting in foliar scorching followed by slow death of the vines. There is no known treatment for Pierce's disease;
Bacterial Spot caused by the bacterium Xanthomonas campestris causes serious disease problems on tomatoes and peppers. It is a significant problem in the Florida tomato industry because it spreads rapidly, especially in warm periods where there is wind-driven rain. Under these conditions, there are no adequate control measures;
Diseases caused by viruses of the family Geminiviridae are a growing agricultural problem worldwide. Geminiviruses have caused severe crop losses in tomato, cassava, and cotton. For instance, in the 1991-1992 growing season in Florida, geminiviruses caused $140 million in damages to the tomato crop (Moffat (1991) Science 286: 1835). Geminiviruses have the ability to recombine between strains to rapidly produce new virulent varieties. Therefore, there is a pressing need for broad-spectrum geminivirus control;
The soybean cyst nematode, Heterodera glycines, causes stunting and chlorosis of soybean plants, which results in yield losses or plant death from severe infestation. Annual losses in the United States have been estimated at $1.5 billion (University of Minnesota Extension Service).
The aforementioned pathogens represent a very small fraction of diverse species that seriously affect plant health and yield. For a more complete description of numerous plant diseases, see, for example, Vidhyasekaran (1997) Fungal Pathogenesis in Plants and Crops: Molecular Biology and Host Defense Mechanisms, Marcel Dekker, Monticello, N.Y.), or Agrios (1997) Plant Pathology, Academic Press, New York, N.Y.). Plants that are able to resist disease may produce significantly higher yields and improved food quality. It is thus of considerable importance to find genes that reduce or prevent disease.
Category: Light Response; Desired Trait: Reduced Shade Avoidance.
Shade avoidance describes the process in which plants grown in close proximity attempt to out-compete each other by increasing stem length at the expense of leaf, fruit and storage organ development. This is caused by the plant's response to far-red radiation reflected from leaves of neighboring plants, which is mediated by phytochrome photoreceptors. Close proximity to other plants, as is produced in high-density crop plantings, increases the relative proportion of far-red irradiation, and therefore induces the shade avoidance response. Shade avoidance adversely affects biomass and yield, particularly when leaves, fruits or other storage organs constitute the desired crop (see, for example, Smith (1982) Annu. Rev. Plant Physiol. 33: 481-518; Ballare et al. (1990) Science 247: 329-332; Smith (1995) Annu. Dev. Plant Physiol. Mol. Biol., 46: 289-315; and Schmitt et al. (1995), American Naturalist, 146: 937-953). Alteration of the shade avoidance response in tobacco through alteration of phytochrome levels has been shown to produce an increase in harvest index (leaf biomass/total biomass) at high planting density, which would result in higher yield (Robson et al. (1996) Nature Biotechnol. 14: 995-998).
Category: Flowering Time; Desired Trait: Altered Flowering Time and Flowering Control.
Timing of flowering has a significant impact on production of agricultural products. For example, varieties with different flowering responses to environmental cues are necessary to adapt crops to different production regions or systems. Such a range of varieties have been developed for many crops, including wheat, corn, soybean, and strawberry. Improved methods for alteration of flowering time will facilitate the development of new, geographically adapted varieties.
Breeding programs for the development of new varieties can be limited by the seed-to-seed cycle. Thus, breeding new varieties of plants with multi-year cycles (such as biennials, e.g. carrot, or fruit trees, such as citrus) can be very slow. With respect to breeding programs, there would be a significant advantage in having commercially valuable plants that exhibit controllable and modified periods to flowering (“flowering times”). For example, accelerated flowering would shorten crop and tree breeding programs.
Improved flowering control allows more than one planting and harvest of a crop to be made within a single season. Early flowering would also improve the time to harvest plants in which the flower portion of the plant constitutes the product (e.g., broccoli, cauliflower, and other edible flowers). In addition, chemical control of flowering through induction or inhibition of flowering in plants could provide a significant advantage to growers by inducing more uniform fruit production (e.g., in strawberry)
A sizable number of plants for which the vegetative portion of the plant forms the valuable crop tend to “bolt” dramatically (e.g., spinach, onions, lettuce), after which biomass production declines and product quality diminishes (e.g., through flowering-triggered senescence of vegetative parts). Delay or prevention of flowering may also reduce or preclude dissemination of pollen from transgenic plants.
Category: Growth Rate; Desired Trait: Modified Growth Rate.
For almost all commercial crops, it is desirable to use plants that establish more quickly, since seedlings and young plants are particularly susceptible to stress conditions such as salinity or disease. Since many weeds may outgrow young crops or out-compete them for nutrients, it would also be desirable to determine means for allowing young crop plants to out compete weed species. Increasing seedling growth rate (emergence) contributes to seedling vigor and allows for crops to be planted earlier in the season with less concern for losses due to environmental factors. Early planting helps add days to the critical grain-filling period and increases yield.
Providing means to speed up or slow down plant growth would also be desirable to ornamental horticulture. If such means be provided, slow growing plants may exhibit prolonged pollen-producing or fruiting period, thus improving fertilization or extending harvesting season.
Category: Growth Rate; Desired Trait: Modified Senescence and Cell Death.
Premature senescence, triggered by various plant stresses, can limit production of both leaf biomass and seed yield. Transcription factor genes that suppress premature senescence or cell death in response to stresses can provide means for increasing yield. Delay of normal developmental senescence could enhance yield also, particularly for those plants for which the vegetative part of the plant represents the commercial product (e.g., spinach, lettuce).
Although leaf senescence is thought to be an evolutionary adaptation to recycle nutrients, the ability to control senescence in an agricultural setting has significant value. For example, a delay in leaf senescence in some maize hybrids is associated with a significant increase in yields and a delay of a few days in the senescence of soybean plants can have a large impact on yield. In an experimental setting, tobacco plants engineered to inhibit leaf senescence had a longer photosynthetic lifespan, and produced a 50% increase in dry weight and seed yield (Gan and Amasino (1995) Science 270: 1986-1988). Delayed flower senescence may generate plants that retain their blossoms longer and this may be of potential interest to the ornamental horticulture industry, and delayed foliar and fruit senescence could improve post-harvest shelf-life of produce.
Further, programmed cell death plays a role in other plant responses, including the resistance response to disease, and some symptoms of diseases, for example, as caused by necrotrophic pathogens such as Botrytis cinerea and Sclerotinia sclerotiorum (Dickman et al. Proc. Natl. Acad. Sci., 98: 6957-6962). Localized senescence and/or cell death can be used by plants to contain the spread of harmful microorganisms. A specific localized cell death response, the “hypersensitive response”, is a component of race-specific disease resistance mediated by plant resistance genes. The hypersensitive response is thought to help limit pathogen growth and to initiate a signal transduction pathway that leads to the induction of systemic plant defenses. Accelerated senescence may be a defense against obligate pathogens, such as powdery mildew, that rely on healthy plant tissue for nutrients. With regard to powdery mildew, Botrytis cinerea and Sclerotinia sclerotiorum and other pathogens, transcription factors that ameliorate cell death and/or damage may reduce the significant economic losses encountered, such as, for example, Botrytis cinerea in strawberry and grape.
Category: Growth Regulator; Desired Trait: Altered Sugar Sensing
Sugars are key regulatory molecules that affect diverse processes in higher plants including germination, growth, flowering, senescence, sugar metabolism and photosynthesis. Sucrose, for example, is the major transport form of photosynthate and its flux through cells has been shown to affect gene expression and alter storage compound accumulation in seeds (source-sink relationships). Glucose-specific hexose-sensing has also been described in plants and is implicated in cell division and repression of “famine” genes (photosynthetic or glyoxylate cycles).
Category: Morphology; Desired Trait: Altered Morphology
Trichomes are branched or unbranched epidermal outgrowths or hair structures on a plant. Trichomes produce a variety of secondary biochemicals such as diterpenes and waxes, the former being important as, for example, insect pheromones, and the latter as protectants against-desiccation and herbivorous pests. Since diterpenes also have commercial value as flavors, aromas, pesticides and cosmetics, and potential value as anti-tumor agents and inflammation-mediating substances, they have been both products and the target of considerable research. In most cases where the metabolic pathways are impossible to engineer, increasing trichome density or size on leaves may be the only way to increase plant productivity. Thus, it would be advantageous to discover trichome-affecting transcription factor genes for the purpose of increasing trichome density, size, or type to produce plants that are better protected from insects or that yield higher amounts of secondary metabolites.
The ability to manipulate wax composition, amount, or distribution could modify plant tolerance to drought and low humidity or resistance to insects, as well as plant appearance. In particular, a possible application for a transcription factor gene that reduces wax production in sunflower seed coats would be to reduce fouling during seed oil processing. Antisense or co-suppression of transcription factors involved in wax biosynthesis in a tissue specific manner can be used to specifically alter wax composition, amount, or distribution in those plants and crops from which wax is either a valuable attribute or product or an undesirable constituent of plants.
Other morphological characteristics that may be desirable in plants include those of an ornamental nature. These include changes in seed color, overall color, leaf and flower shape, leaf color, leaf size, or glossiness of leaves. Plants that produce dark leaves may have benefits for human health; flavonoids, for example, have been used to inhibit tumor growth, prevent of bone loss, and prevention lipid oxidation in animals and humans. Plants in which leaf size is increased would likely provide greater biomass, which would be particularly valuable for crops in which the vegetative portion of the plant constitutes the product. Plants with glossy leaves generally produce greater epidermal wax, which, if it could be augmented, resulted in a pleasing appearance for many ornamentals, help prevent desiccation, and resist herbivorous insects and disease-causing agents. Changes in plant or plant part coloration, brought about by modifying, for example, anthocyanin levels, would provide novel morphological features.
In many instances, the seeds of a plant constitute a valuable crop. These include, for example, the seeds of many legumes, nuts and grains. The discovery of means for producing larger seed would provide significant value by bringing about an increase in crop yield.
Plants with altered inflorescence, including, for example, larger flowers or distinctive floral configurations, may have high value in the ornamental horticulture industry.
Modifications to flower structure may have advantageous or deleterious effects on fertility, and could be used, for example, to decrease fertility by the absence, reduction or screening of reproductive components. This could be a desirable trait, as it could be exploited to prevent or minimize the escape of the pollen of genetically modified organisms into the environment.
Manipulation of inflorescence branching patterns may also be used to influence yield and offer the potential for more effective harvesting techniques. For example, a “self pruning” mutation of tomato results in a determinate growth pattern and facilitates mechanical harvesting (Pnueli et al. (2001) Plant Cell 13(12): 2687-2702).
Alterations of apical dominance or plant architecture could create new plant varieties. Dwarf plants may be of potential interest to the ornamental horticulture industry.
Category: Seed Biochemistry; Desired Trait: Altered Seed Oil
The composition of seeds, particularly with respect to seed oil quantity and/or composition, is very important for the nutritional value and production of various food and feed products. Desirable improvements to oils include enhanced heat stability, improved nutritional quality through, for example, reducing the number of calories in seed, increasing the number of calories in animal feeds, or altering the ratio of saturated to unsaturated lipids comprising the oils.
Category: Seed Biochemistry; Desired Trait: Altered Seed Protein
As with seed oils, seed protein content and composition is very important for the nutritional value and production of various food and feed products. Altered protein content or concentration in seeds may be used to provide nutritional benefits, and may also prolong storage capacity, increase seed pest or disease resistance, or modify germination rates. Altered amino acid composition of seeds, through altered protein composition, is also a desired objective for nutritional improvement.
Category: Seed Biochemistry; Desired Trait: Altered Prenyl Lipids.
Prenyl lipids, including the tocopherols, play a role in anchoring proteins in membranes or membranous organelles. Tocopherols have both anti-oxidant and vitamin E activity. Modified tocopherol composition of plants may thus be useful in improving membrane integrity and function, which may mitigate abiotic stresses such as heat stress. Increasing the anti-oxidant and vitamin content of plants through increased tocopherol content can provide useful human health benefits.
Category: Leaf Biochemistry; Desired Trait: Altered Glucosinolate Levels
Increases or decreases in specific glucosinolates or total glucosinolate content can be desirable depending upon the particular application. For example: (i) glucosinolates are undesirable components of the oilseeds used in animal feed, since they produce toxic effects; low-glucosinolate varieties of canola have been developed to combat this problem; (ii) some glucosinolates have anti-cancer activity; thus, increasing the levels or composition of these compounds can be of use in production of nutraceuticals; and (iii) glucosinolates form part of a plant's natural defense against insects; modification of glucosinolate composition or quantity could therefore afford increased protection from herbivores. Furthermore, tissue specific promoters can be used in edible crops to ensure that these compounds accumulate specifically in particular tissues, such as the epidermis, which are not taken for human consumption.
Category: Leaf Biochemistry; Desired Trait: Flavonoid Production.
Expression of transcription factors that increase flavonoid production in plants, including anthocyanins and condensed tannins, may be used to alter pigment production for horticultural purposes, and possibly to increase stress resistance. Flavonoids have antimicrobial activity and could be used to engineer pathogen resistance. Several flavonoid compounds have human health promoting effects such as inhibition of tumor growth, prevention of bone loss and prevention of lipid oxidation. Increased levels of condensed tannins in forage legumes would provide agronomic benefits in ruminants by preventing pasture bloat by collapsing protein foams within the rumen. For a review on the utilities of flavonoids and their derivatives, see Dixon et al. (1999) Trends Plant Sci. 4: 394-400.
Genetic and molecular studies on Arabidopsis have revealed that the timing of flowering is influenced by a large number of different genes (Martinez-Zapater and Somerville (1990) Plant Physiol. 92: 770-776; Koornneef et al. (1991) Mol. Gen. Genet. 229: 57-66; Martinez-Zapater et al. (1994) In Meyerowitz and Somerville, editors, Arabidopsis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 403-433; Koornneef et al. (1998a) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 345-370; Koornneef et al. (1998b) Genetics 148: 885-892; Levy and Dean (1998) Plant Cell 10: 1973-1990; Simpson et al. (1999) Annu. Rev. Cell Dev. Biol. 15: 519-550; Simpson and Dean (2002) Science 296: 285-289; and Ratcliffe and Riechmann (2002) Curr. Issues Mol. Biol. 4: 77-91). Such loci ensure that the switch from vegetative to reproductive growth takes place at the most appropriate time with respect to a variety of abiotic and biotic variables. Amongst the most intensively studied effects are the responses to day length and prolonged exposure to low temperatures (vernalization).
Arabidopsis flowers rapidly in long day photoperiodic conditions of 16 hours or continuous light. However, under short day conditions of 8-10 hours of light, the plants display a much more extensive period of vegetative growth prior to flowering. Genes that control this day length response were originally identified via mutations that cause late flowering under long days, but which do not alter flowering time in short day conditions. Examples of photoperiod pathway genes include CONSTANS (CO), GIGANTEA (GI), FE, FD, and FHA. A second group of genes, which includes LUMINIDEPENDENS (LD), FCA, FVE, FY, and FPA, form an autonomous pathway that monitors the developmental state of the plant and is active under all photoperiodic conditions. Mutants for this second class of genes flower later than wild type controls irrespective of the day length (Koornneef et al. (1991); Martinez-Zapater et al. (1994); Koornneef et al. (1998a); and Koornneef et al. (1998b); all supra).
Importantly, mutants from the photoperiod and autonomous pathways also show a differential response to vernalization. Via a vernalization response, Arabidopsis ecotypes from northern latitudes, such as Stockholm, Sweden, are adapted to flower in the spring following exposure to cold winter conditions. This avoids flowering in the late summer when seed maturation might be curtailed by the onset of winter conditions. (See, for example, Vince-Prue (1975) In Photoperiodism in plants McGraw Hill, London, UK, pp 263-291; Napp-Zinn (1957) Z. Indukt. Abstammungs Vererbungsl. 88: 253-285; and Reeves and Coupland (2000) Curr. Opin. Plant Biol. 3: 37-42).
When such ecotypes are grown in the laboratory they flower late, but will flower much earlier if subjected to a cold period of 4-8 weeks while the seed is germinating. In a comparable manner, mutants from the autonomous pathway exhibit a very marked reduction in flowering time when subjected to vernalization. By contrast, mutants from the photoperiod pathway show only a minor response to cold treatments. Thus, vernalization can overcome the requirement for the autonomous pathway. (See Martinez-Zapater and Somerville (1990) supra; Koornneef et al. (1991) supra; Bagnall (1992) Aust. J. Plant Physiol. 19: 401-409; Burn et al. (1993) Proc. Natl. Acad. Sci. 90: 287-291; Lee et al. (1993) Mol. Gen. Genet. 237: 171-176; Clarke and Dean (1994) Mol. Gen. Genet. 242: 81-89; Chandler et al. (1996) Plant J. 10: 637-644; Koornneef et al. (1998b) supra.)
Genetic and molecular analyses have revealed that a MADS box protein, FLOWERING LOCUS C (FLC), is a major determinant of the vernalization response (Koornneef et al. (1994) supra; Lee et al. (1994) supra; Sanda and Amasino (1996) Mol. Gen. Genet. 251: 69-74; Michaels and Amasino (2000) Plant Cell and Environment 23: 1145-1153; Sheldon et al. (1999) Plant Cell 11: 445-458; Sheldon et al. (2002) Plant Cell 14: 2527-2537; and Rouse et al. (2002) Plant J. 29: 183-191). High levels of both FLC gene transcript and protein are present in mutants for the autonomous pathway and also in naturally late flowering northern ecotypes, which contain active alleles of a second locus, FRIGIDA (FR1; Burn et al. (1993) supra; Clarke and Dean (1994) supra; Johanson et al. (2000) Science 290: 344-347). By contrast, mutants from the photoperiod pathway, and backgrounds lacking an active FRI allele, show relatively low levels of FLC transcript. Furthermore, null alleles of flc completely suppress the late flowering caused by autonomous pathway mutations and active FRI alleles, but have no effect on the delayed flowering in photoperiod pathway mutants (Michaels and Amasino (2001) Plant Cell 13: 935-941). FLC gene expression therefore appears to be supported by FRI and strongly repressed by floral activators within the autonomous pathway.
During vernalization, FLC transcript and protein levels fall, and the plants become competent to flower (Michaels and Amasino (1999) Plant Cell 11: 949-956; Michaels and Amasino (2001) supra; Sheldon et al. (1999) supra; Sheldon et al. (2000) Proc. Natl. Acad. Sci. 97: 3753-3758; Johanson et al. (2000) supra; and Rouse et al. (2002) supra). Additionally, overexpression of FLC from a 35S CaMV promoter in the Landsberg ecotype (which lacks an active FRI allele) is sufficient to severely delay or prevent flowering, and renders the plants insensitive to vernalization (Michaels and Amasino (1999) supra; Sheldon et al. (1999) supra). These findings indicate that FLC is a potent floral repressor; it has now been shown that such repression is achieved by FLC inhibiting downstream genes that promote flowering, including SOCI and FT (Borner et al. (2000) Plant J. 24: 591-599; Lee et al. (2000) Genes Dev. 14: 2366-2376; Onouchi et al. (2000) Plant Cell 12: 885-900; Samach et al. (2000) Science 288: 1613-1616; Michaels and Amasino (2001) supra). Thus, promotion of flowering by either the autonomous pathway or vernalization involves repression of FLC and the subsequent de-repression of FLC targets. Recently, regions within the FLC gene and its promoter have been defined which are required for its vernalization induced repression (Sheldon et al. (2002) supra). However, the molecular signaling events that lead to a fall in FLC levels during vernalization are still unclear. The products of VERNALIZATION2 and VERNALIZATION1 maintain repression of FLC, once levels of FLC transcript have declined (Gendall et al. (2001) Cell 107: 525-535; Levy et al. (2002) Science 297: 243-246), but it is not yet known how the decline is initially achieved.
A number of additional questions, regarding the molecular basis of vernalization, still remain unanswered. First, it has been observed that null flc mutants are responsive to vernalization (Michaels and Amasino (2001) supra). Therefore vernalization can promote flowering by other mechanisms as well as via repression of FLC. In addition, vernalization is a quantitative response to prolonged periods of cold (Sheldon et al. (2000) supra); a mechanism must therefore exist to ensure that vernalization does not always occur in response to short periods of cold, lasting only a few days.
Vernalization may also be desirable in plants that do not normally have a vernalization response. Such plants in which expression of a polynucleotide creates a vernalization response therefore may be propagated and cultivated at different latitudes and/or altitudes compared with the native plant species that do not express a polynucleotide creating a vernalization response.
The present invention relates to methods and compositions for producing transgenic plants with modified traits, particularly traits that address the agricultural and food needs described in the above background information. These traits may provide significant value in that they allow the plant to thrive in hostile environments, where, for example, temperature, water and nutrient availability or salinity may limit or prevent growth of non-transgenic plants. The traits may also comprise desirable morphological alterations, larger or smaller size, disease and pest resistance, alterations in flowering time, light response, and others.
We have identified polynucleotides encoding transcription factors, developed numerous transgenic plants using these polynucleotides, and have analyzed the plants for a variety of important traits. In so doing, we have identified important polynucleotide and polypeptide sequences for producing commercially valuable plants and crops as well as the methods for making them and using them. Other aspects and embodiments of the invention are described below and can be derived from the teachings of this disclosure as a whole.